Determination of antidepressants by mass spectrometry

ABSTRACT

Methods are described for detecting or determining the amount of antidepressants and/or antidepressant metabolites in a sample. More specifically, mass spectrometric methods are described for detecting and quantifying antidepressants and/or antidepressant metabolites in a sample.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application claims benefit of U.S. Provisional Application No.62/855,863, filed May 31, 2019, which is incorporated by referenceherein in its entirety.

BACKGROUND OF THE INVENTION

Baseline testing is useful to help clinicians determine patient use ornon-use of an antidepressant drug or drugs prior to treatment. It iscrucial to monitor patients who are prescribed antidepressants to ensurecompliance and avoid unintended polydrug use. Some antidepressants, suchas selective serotonin reuptake inhibitors (SSRIs), may have harmfulside effects and should not be mixed with drugs within the same class.An accurate testing for antidepressant and metabolites is needed.

SUMMARY OF THE INVENTION

In one aspect, provided herein are methods for detection andquantitation of antidepressants and antidepressant metabolites by massspectrometry.

Provided herein are methods for detecting the presence or amount ofantidepressants and/or antidepressant metabolites in a sample by massspectrometry. The methods include subjecting the sample to ionizationunder conditions suitable to produce one or more ions detectable by massspectrometry; determining the amount of one or more ions by massspectrometry; and using the amount of one or more ions to determine thepresence or amount of antidepressants and/or antidepressant metabolitesin the sample.

In some embodiments, mass spectrometry comprises tandem massspectrometry. In these embodiments, the methods include: a) ionizing thesample under conditions suitable to produce a precursor ion; b)fragmenting a precursor ion to produce one or more fragment ions; c)determining the amount of one or more ions produced in steps a) and b);and d) using the amount of the one or more ions determined in step c) todetermine the presence or amount of antidepressants and metabolites inthe sample.

In some embodiments, provided herein are methods for detecting ordetermining the amount of one or more antidepressants and antidepressantmetabolites comprising selective serotonin reuptake inhibitors,serotonin and norepinephrine reuptake inhibitors, norepinephrine anddopamine reuptake inhibitors, tricyclic antidepressants, sedatives,and/or antidepressant metabolites metabolites.

In some embodiments, provided herein are methods for detecting ordetermining the amount of one or more antidepressants and antidepressantmetabolites selected from the group consisting of fluoxetine,paroxetine, sertraline, citalopram, escitalopram, fluvoxamine,vilazodone, duloxetine, venlafaxine, desmethylvenlafaxine,hydroxybupropion, imipramine, nortriptyline, amitriptyline, doxepin,trimipramine, desipramine, protriptyline, amoxapine, clomipramine,maprotiline, trazodone, mirtazapine, vortioxetine, desmethylcitalopram,desmethylclomipramine, desmethyldoxepin, norfluoxetine, norfluvoxamine,norsertraline, and 1,3-chlorphenylpiperazine.

In some embodiments, provided herein are methods for detecting ordetermining the amount of one or more selective serotonin reuptakeinhibitors (fluoxetine, paroxetine, sertraline, citalopram,escitalopram, fluvoxamine, vilazodone); serotonin and norepinephrinereuptake inhibitors (duloxetine, venlafaxine, desmethylvenlafaxine);norepinephrine and dopamine reuptake inhibitors (hydroxybupropion);tricyclic antidepressants (imipramine, nortriptyline, amitriptyline,doxepin, trimipramine, desipramine, protriptyline, amoxapine,clomipramine, maprotiline). Other antidepressants used in this assayalso act as sedatives and are trazodone, mirtazapine and vortioxetine.Metabolites tested were desmethylcitalopram, desmethylclomipramine,desmethyldoxepin, norfluoxetine, norfluvoxamine, norsertraline, and1,3-chlorphenylpiperazine.

In some embodiments, provided herein are methods for simultaneouslydetecting or determining the amount of 10 or more antidepressants andantidepressant metabolites.

In some embodiments, provided herein are methods for simultaneouslydetecting or determining the amount of 20 or more antidepressants andantidepressant metabolites.

In some embodiments, provided herein are methods for simultaneouslydetecting or determining the amount of 30 antidepressants andantidepressant metabolites.

In some embodiments, the methods provided herein comprise adding one ormore internal standards. In some embodiments, the one or more internalstandards comprise deuterated internal standards. In some embodiments,the deuterated internal standards are selected from the group consistingof 1,3-chlorphenylpiperazine-D8, hydroxybupropion-D6,desmethyl-venlafaxine-D6, desmethylcitalopram-D3, trimipramine-D3,amitriptyline-D3, nortriptyline-D3, paroxetine-D6, protriptyline-D3,citalopram-D6, venlafaxine-D6, imipramine-D3, trazodone-D6,vilazodone-D4, and vortioxetine-D8.

In some embodiments, the sample comprises a biological sample. In apreferred embodiment, the sample is urine. In some embodiments, thesample is plasma or serum. In some embodiments, the sample is blood.

In some embodiments, the sample is subjected to liquid chromatographyprior to ionization. In some embodiments, the liquid chromatographycomprises high performance liquid chromatography.

In some embodiments, the method is capable of detecting antidepressantsand antidepressant metabolites at levels within the range of about 4ng/mL to about 5000 ng/mL, inclusive.

In some embodiments, the method is capable of detecting antidepressantsand antidepressant metabolites at levels within the range of about 25ng/mL to about 5000 ng/mL, inclusive.

In some embodiments, the mass spectrometry is tandem mass spectrometry.In some embodiments, the tandem mass spectrometry is conducted byselected reaction monitoring, multiple reaction monitoring, precursorion scanning, or product ion scanning.

In a preferred embodiment, the tandem mass spectrometry is conducted byselected reaction monitoring.

In some embodiments, provided herein are determining the antidepressantsand antidepressant metabolites comprising detecting ions comprising thefollowing mass/charge ratios (m/z).

Precursor Fragment Q1 (m/z) Q3 (m/z) Analyte  1 196.993 1181,3-Chlorphenylpiperazine 1  2 196.993 119.1 1,3-Chlorphenylpiperazine 2 3 205.065 158.1 1,3-Chlorphenylpiperazine D8  4 278.096 105Amitriptyline 1  5 278.096 115 Amitriptyline 2  6 281 202.1Amitriptyline-D3  7 314.006 271.1 Amoxapine 1  8 314.006 193.1 Amoxapine2  9 256.02 130 Hydroxybupropion 1 10 256.02 103 Hydroxybupropion 2 11262.061 130.1 Hydroxybupropion-D6 12 325.07 109 Citalopram 1 13 325.07262.1 Citalopram 2 14 331.103 109 Citalopram-D6 15 264.083 91Nortriptyline 1 16 264.083 105 Nortriptyline 2 17 267.095 105Nortriptyline-D3 18 311.043 109 Desmethylcitalopram 1 19 311.043 262.1Desmethylcitalopram 2 20 314.072 108.9 Desmethylcitalopram-D3 21 315.05486.1 Clomipramine 1 22 315.054 58 Clomipramine 2 23 301.037 72Desmethylclomipramine 1 24 301.037 227.1 Desmethylclomipramine 2 25267.091 72 Desipramine 1 26 267.091 193.1 Desipramine 2 27 280.096 107Doxepin 1 28 280.096 165.1 Doxepin 2 29 266.074 107 Desmethyldoxapin 130 266.074 77 Desmethyldoxapin 2 31 298.03 154.1 Duloxetine 1 32 298.0344.1 Duloxetine 2 33 310.07 148.1 Fluoxetine 1 34 310.07 44.1 Fluoxetine2 35 296.066 134.2 Norfluoxetine 1 36 296.066 30.1 Norfluoxetine 2 37319.057 71 Fluvoxamine 1 38 319.057 200.1 Fluvoxamine 2 39 305.025 229.1Norfluvoxamine 1 40 305.025 188.1 Norfluvoxamine 2 41 281.098 86Imipramine 1 42 281.098 58 Imipramine 2 43 284.013 89 Imipramine-D3 44278.094 191.2 Maprotiline 1 45 278.094 189 Maprotiline 2 46 266.081195.1 Mirtazapine 1 47 266.081 194.1 Mirtazapine 2 48 330.033 192.1Paroxetine 1 49 330.033 70 Paroxetine 2 50 336.092 198.2 Paroxetine-D651 264.096 191 Protriptyline 1 52 264.096 189 Protriptyline 2 53 267.095191.1 Protriptyline-D3 54 306 159 Sertraline 1 55 306 275 Sertraline 256 292.005 159 Desmethylsertraline 1 57 292.005 123 Desmethylsertraline2 58 372.096 176.1 Trazodone 1 59 372.096 148 Trazodone 2 60 378.114182.1 Trazodone-D6 61 295.128 100.1 Trimipramine 1 62 295.128 58.1Trimipramine 2 63 298.138 103.1 Trimipramine-D3 64 278.126 58Venlafaxine 1 65 278.126 121 Venlafaxine 2 66 284.139 64.1Venlafaxine-D6 67 264.106 58 Desmethylvenlafaxine 1 68 264.106 107Desmethylvenlafaxine 2 69 270.134 64.1 Desmethylvenlafaxine-D6 70442.133 155.1 Vilazodone 1 71 442.133 197.2 Vilazodone 2 72 446.163155.1 Vilazodone-D4 73 299.059 150 Vortioxetine 1 74 299.059 109Vortioxetine 2 75 307.082 153.1 Vortioxetine-D8

In some embodiments, the methods described herein are capable ofdetecting antidepressants and antidepressant metabolites at levelswithin the range of 4 ng/mL to 5000 ng/mL, inclusive. In someembodiments, the methods described herein are capable of detectingantidepressants and antidepressant metabolites at levels within therange of 25 ng/mL to 5000 ng/mL, inclusive.

In some embodiments, the methods described herein are capable ofquantitating antidepressants and antidepressant metabolites at lowerlimit of 10 ng/mL. In some embodiments, the methods described herein arecapable of quantitating antidepressants and antidepressant metabolitesat lower limit of 50 ng/mL.

In some embodiments, the sample is subjected to an extraction column,such as a solid phase extraction (SPE) column, prior to ionization. Insome related embodiments, SPE and mass spectrometry are conducted withon-line processing.

In some embodiments, the sample is subjected to an analytical column,such as a high performance liquid chromatography (HPLC) column, prior toionization. In some related embodiments, HPLC and mass spectrometry areconducted with on-line processing.

In some embodiments, the methods may be used to determine the presenceor amount of antidepressants and antidepressant metabolites in abiological sample; such as plasma or serum. In some related embodiments,a biological sample is processed by one or more steps to generate aprocessed sample, which may then be subjected to mass spectrometricanalysis. In some embodiments, the one or more processing steps compriseone or more purification steps, such as protein precipitation,filtration, liquid-liquid extraction, solid phase extraction, liquidchromatography, any immunopurification process, or the like, and anycombination thereof.

In certain preferred embodiments of the methods disclosed herein, massspectrometry is performed in positive ion mode. Alternatively, massspectrometry is performed in negative ion mode. Various ionizationsources, including for example atmospheric pressure chemical ionization(APCI) or electrospray ionization (ESI), may be used in embodiments ofthe present invention. In certain embodiments, antidepressants andantidepressant metabolites are measured using positive ion mode.

In preferred embodiments, a separately detectable internal standard isprovided in the sample, the amount of which is also determined in thesample. In these embodiments, all or a portion of both the analyte ofinterest and the internal standard present in the sample is ionized toproduce a plurality of ions detectable in a mass spectrometer, and oneor more ions produced from each are detected by mass spectrometry. Inthese embodiments, the presence or amount of ions generated from theanalyte of interest may be related to the presence of amount of analyteof interest in the sample.

In other embodiments, the amount of the antidepressants andantidepressant metabolites in a sample may be determined by comparisonto one or more external reference standards. Exemplary externalreference standards include blank plasma or serum spiked withantidepressants and antidepressant metabolites or an isotopicallylabeled variant thereof.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to “aprotein” includes a plurality of protein molecules.

As used herein, the term “purification” or “purifying” does not refer toremoving all materials from the sample other than the analyte(s) ofinterest. Instead, purification refers to a procedure that enriches theamount of one or more analytes of interest relative to other componentsin the sample that may interfere with detection of the analyte ofinterest. Purification of the sample by various means may allow relativereduction of one or more interfering substances, e.g., one or moresubstances that may or may not interfere with the detection of selectedparent or daughter ions by mass spectrometry. Relative reduction as thisterm is used does not require that any substance, present with theanalyte of interest in the material to be purified, is entirely removedby purification.

As used herein, the term “immunopurification” or “immunopurify” refersto a purification procedure that utilizes antibodies, includingpolyclonal or monoclonal antibodies, to enrich the one or more analytesof interest. Immunopurification can be performed using any of theimmunopurification methods well known in the art. Often theimmunopurification procedure utilizes antibodies bound, conjugated orotherwise attached to a solid support, for example a column, well, tube,gel, capsule, particle or the like. Immunopurification as used hereinincludes without limitation procedures often referred to in the art asimmunoprecipitation, as well as procedures often referred to in the artas affinity chromatography.

As used herein, the term “immunoparticle” refers to a capsule, bead, gelparticle or the like that has antibodies bound, conjugated or otherwiseattached to its surface (either on and/or in the particle). In certainembodiments utilizing immunopurification, immunoparticles comprisesepharose or agarose beads. In alternative embodiments utilizingimmunopurification, immunoparticles comprise glass, plastic or silicabeads, or silica gel.

As used herein, the term “sample” refers to any sample that may containan analyte of interest. As used herein, the term “body fluid” means anyfluid that can be isolated from the body of an individual. For example,“body fluid” may include blood, plasma, serum, bile, saliva, urine,tears, perspiration, and the like. In some embodiments, the samplecomprises a body fluid sample; preferably plasma or serum.

As used herein, the term “solid phase extraction” or “SPE” refers to aprocess in which a chemical mixture is separated into components as aresult of an affinity of components dissolved or suspended in a solution(i.e., mobile phase) for a solid through or around which the solution ispassed (i.e., solid phase). SPE, as used herein, is distinct fromimmunopurification in that the affinity of components in the mobilephase to the solid phase is the result of a chemical or physicalinteraction, rather than an immunoaffinity. In some instances, as themobile phase passes through or around the solid phase, undesiredcomponents of the mobile phase may be retained by the solid phaseresulting in a purification of the analyte in the mobile phase. In otherinstances, the analyte may be retained by the solid phase, allowingundesired components of the mobile phase to pass through or around thesolid phase. In these instances, a second mobile phase is then used toelute the retained analyte off of the solid phase for further processingor analysis. SPE, including TFLC, may operate via a unitary or mixedmode mechanism. Mixed mode mechanisms utilize ion exchange andhydrophobic retention in the same column; for example, the solid phaseof a mixed-mode SPE column may exhibit strong anion exchange andhydrophobic retention; or may exhibit column exhibit strong cationexchange and hydrophobic retention.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas is separated into componentsas a result of differential distribution of the chemical entities asthey flow around or over a stationary liquid or solid phase.

As used herein, the term “liquid chromatography” or “LC” means a processof selective retardation of one or more components of a fluid solutionas the fluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retardation resultsfrom the distribution of the components of the mixture between one ormore stationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). Examples of “liquidchromatography” include reverse phase liquid chromatography (RPLC), highperformance liquid chromatography (HPLC), and turbulent flow liquidchromatography (TFLC) (sometimes known as high turbulence liquidchromatography (HTLC) or high throughput liquid chromatography).

As used herein, the term “high performance liquid chromatography” or“HPLC” (sometimes known as “high pressure liquid chromatography”) refersto liquid chromatography in which the degree of separation is increasedby forcing the mobile phase under pressure through a stationary phase,typically a densely packed column.

As used herein, the term “turbulent flow liquid chromatography” or“TFLC” (sometimes known as high turbulence liquid chromatography or highthroughput liquid chromatography) refers to a form of chromatographythat utilizes turbulent flow of the material being assayed through thecolumn packing as the basis for performing the separation. TFLC has beenapplied in the preparation of samples containing two unnamed drugs priorto analysis by mass spectrometry. See, e.g., Zimmer et al., J ChromatogrA 854: 23-35 (1999); see also, U.S. Pat. Nos. 5,968,367, 5,919,368,5,795,469, and 5,772,874, which further explain TFLC. Persons ofordinary skill in the art understand “turbulent flow”. When fluid flowsslowly and smoothly, the flow is called “laminar flow”. For example,fluid moving through an HPLC column at low flow rates is laminar. Inlaminar flow the motion of the particles of fluid is orderly withparticles moving generally in straight lines. At faster velocities, theinertia of the water overcomes fluid frictional forces and turbulentflow results. Fluid not in contact with the irregular boundary “outruns”that which is slowed by friction or deflected by an uneven surface. Whena fluid is flowing turbulently, it flows in eddies and whirls (orvortices), with more “drag” than when the flow is laminar. Manyreferences are available for assisting in determining when fluid flow islaminar or turbulent (e.g., Turbulent Flow Analysis: Measurement andPrediction, P. S. Bernard & J. M. Wallace, John Wiley & Sons, Inc.,(2000); An Introduction to Turbulent Flow, Jean Mathieu & Julian Scott,Cambridge University Press (2001)).

As used herein, the term “gas chromatography” or “GC” refers tochromatography in which the sample mixture is vaporized and injectedinto a stream of carrier gas (as nitrogen or helium) moving through acolumn containing a stationary phase composed of a liquid or aparticulate solid and is separated into its component compoundsaccording to the affinity of the compounds for the stationary phase.

As used herein, the term “large particle column” or “extraction column”refers to a chromatography column containing an average particlediameter greater than about 50 μm. As used in this context, the term“about” means ±10%.

As used herein, the term “analytical column” refers to a chromatographycolumn having sufficient chromatographic plates to effect a separationof materials in a sample that elute from the column sufficient to allowa determination of the presence or amount of an analyte. Such columnsare often distinguished from “extraction columns”, which have thegeneral purpose of separating or extracting retained material fromnon-retained materials in order to obtain a purified sample for furtheranalysis. As used in this context, the term “about” means ±10%. In apreferred embodiment the analytical column contains particles of about 5μm in diameter.

As used herein, the terms “on-line” and “inline”, for example as used in“on-line automated fashion” or “on-line extraction” refers to aprocedure performed without the need for operator intervention. Incontrast, the term “off-line” as used herein refers to a procedurerequiring manual intervention of an operator. Thus, if samples aresubjected to precipitation, and the supernatants are then manuallyloaded into an autosampler, the precipitation and loading steps areoff-line from the subsequent steps. In various embodiments of themethods, one or more steps may be performed in an on-line automatedfashion.

As used herein, the term “mass spectrometry” or “MS” refers to ananalytical technique to identify compounds by their mass. MS refers tomethods of filtering, detecting, and measuring ions based on theirmass-to-charge ratio, or “m/z”. MS technology generally includes (1)ionizing the compounds to form charged compounds; and (2) detecting themolecular weight of the charged compounds and calculating amass-to-charge ratio. The compounds may be ionized and detected by anysuitable means. A “mass spectrometer” generally includes an ionizer andan ion detector. In general, one or more molecules of interest areionized, and the ions are subsequently introduced into a massspectrometric instrument where, due to a combination of magnetic andelectric fields, the ions follow a path in space that is dependent uponmass (“m”) and charge (“z”). See, e.g., U.S. Pat. No. 6,204,500,entitled “Mass Spectrometry From Surfaces;” U.S. Pat. No. 6,107,623,entitled “Methods and Apparatus for Tandem Mass Spectrometry;” U.S. Pat.No. 6,268,144, entitled “DNA Diagnostics Based On Mass Spectrometry;”U.S. Pat. No. 6,124,137, entitled “Surface-Enhanced PhotolabileAttachment And Release For Desorption And Detection Of Analytes;” Wrightet al., Prostate Cancer and Prostatic Diseases 1999, 2: 264-76; andMerchant and Weinberger, Electrophoresis 2000, 21: 1164-67.

As used herein, the term “operating in negative ion mode” refers tothose mass spectrometry methods where negative ions are generated anddetected. The term “operating in positive ion mode” as used herein,refers to those mass spectrometry methods where positive ions aregenerated and detected.

As used herein, the term “ionization” or “ionizing” refers to theprocess of generating an analyte ion having a net electrical chargeequal to one or more electron units. Negative ions are those having anet negative charge of one or more electron units, while positive ionsare those having a net positive charge of one or more electron units.

As used herein, the term “electron ionization” or “EI” refers to methodsin which an analyte of interest in a gaseous or vapor phase interactswith a flow of electrons. Impact of the electrons with the analyteproduces analyte ions, which may then be subjected to a massspectrometry technique.

As used herein, the term “chemical ionization” or “CI” refers to methodsin which a reagent gas (e.g. ammonia) is subjected to electron impact,and analyte ions are formed by the interaction of reagent gas ions andanalyte molecules.

As used herein, the term “fast atom bombardment” or “FAB” refers tomethods in which a beam of high energy atoms (often Xe or Ar) impacts anon-volatile sample, desorbing and ionizing molecules contained in thesample. Test samples are dissolved in a viscous liquid matrix such asglycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether,2-nitrophenyloctyl ether, sulfolane, diethanolamine, andtriethanolamine. The choice of an appropriate matrix for a compound orsample is an empirical process.

As used herein, the term “matrix-assisted laser desorption ionization”or “MALDI” refers to methods in which a non-volatile sample is exposedto laser irradiation, which desorbs and ionizes analytes in the sampleby various ionization pathways, including photo-ionization, protonation,deprotonation, and cluster decay. For MALDI, the sample is mixed with anenergy-absorbing matrix, which facilitates desorption of analytemolecules.

As used herein, the term “surface enhanced laser desorption ionization”or “SELDI” refers to another method in which a non-volatile sample isexposed to laser irradiation, which desorbs and ionizes analytes in thesample by various ionization pathways, including photo-ionization,protonation, deprotonation, and cluster decay. For SELDI, the sample istypically bound to a surface that preferentially retains one or moreanalytes of interest. As in MALDI, this process may also employ anenergy-absorbing material to facilitate ionization.

As used herein, the term “electrospray ionization” or “ESI,” refers tomethods in which a solution is passed along a short length of capillarytube, to the end of which is applied a high positive or negativeelectric potential. Solution reaching the end of the tube is vaporized(nebulized) into a jet or spray of very small droplets of solution insolvent vapor. This mist of droplets flows through an evaporationchamber. As the droplets get smaller the electrical surface chargedensity increases until such time that the natural repulsion betweenlike charges causes ions as well as neutral molecules to be released.

As used herein, the term “atmospheric pressure chemical ionization” or“APCI,” refers to mass spectrometry methods that are similar to ESI;however, APCI produces ions by ion-molecule reactions that occur withina plasma at atmospheric pressure. The plasma is maintained by anelectric discharge between the spray capillary and a counter electrode.Then ions are typically extracted into the mass analyzer by use of a setof differentially pumped skimmer stages. A counterflow of dry andpreheated N₂ gas may be used to improve removal of solvent. Thegas-phase ionization in APCI can be more effective than ESI foranalyzing less-polar species.

The term “atmospheric pressure photoionization” or “APPI” as used hereinrefers to the form of mass spectrometry where the mechanism for thephotoionization of molecule M is photon absorption and electron ejectionto form the molecular ion M+. Because the photon energy typically isjust above the ionization potential, the molecular ion is lesssusceptible to dissociation. In many cases it may be possible to analyzesamples without the need for chromatography, thus saving significanttime and expense. In the presence of water vapor or protic solvents, themolecular ion can extract H to form MH+. This tends to occur if M has ahigh proton affinity. This does not affect quantitation accuracy becausethe sum of M+ and MH+ is constant. Drug compounds in protic solvents areusually observed as MH+, whereas nonpolar compounds such as naphthaleneor testosterone usually form M+. See, e.g., Robb et al., Anal. Chem.2000, 72(15): 3653-3659.

As used herein, the term “inductively coupled plasma” or “ICP” refers tomethods in which a sample interacts with a partially ionized gas at asufficiently high temperature such that most elements are atomized andionized.

As used herein, the term “field desorption” refers to methods in which anon-volatile test sample is placed on an ionization surface, and anintense electric field is used to generate analyte ions.

As used herein, the term “desorption” refers to the removal of ananalyte from a surface and/or the entry of an analyte into a gaseousphase. Laser desorption thermal desorption is a technique wherein asample containing the analyte is thermally desorbed into the gas phaseby a laser pulse. The laser hits the back of a specially made 96-wellplate with a metal base. The laser pulse heats the base and the heatcauses the sample to transfer into the gas phase. The gas phase sampleis then drawn into the mass spectrometer.

As used herein, the term “selective ion monitoring” is a detection modefor a mass spectrometric instrument in which only ions within arelatively narrow mass range, typically about one mass unit, aredetected.

As used herein, “multiple reaction mode,” sometimes known as “selectedreaction monitoring,” is a detection mode for a mass spectrometricinstrument in which a precursor ion and one or more fragment ions areselectively detected.

As used herein, the term “lower limit of quantification”, “lower limitof quantitation” or “LLOQ” refers to the point where measurements becomequantitatively meaningful. The analyte response at this LOQ isidentifiable, discrete and reproducible with a relative standarddeviation (RSD %) of less than 20% and an accuracy of 85% to 115%.

As used herein, the term “limit of detection” or “LOD” is the point atwhich the measured value is larger than the uncertainty associated withit. The LOD is the point at which a value is beyond the uncertaintyassociated with its measurement and is defined as three times the RSD ofthe mean at the zero concentration.

As used herein, an “amount” of an analyte in a body fluid sample refersgenerally to an absolute value reflecting the mass of the analytedetectable in volume of sample. However, an amount also contemplates arelative amount in comparison to another analyte amount. For example, anamount of an analyte in a sample can be an amount which is greater thana control or normal level of the analyte normally present in the sample.

The term “about” as used herein in reference to quantitativemeasurements not including the measurement of the mass of an ion, refersto the indicated value plus or minus 10%. Mass spectrometry instrumentscan vary slightly in determining the mass of a given analyte. The term“about” in the context of the mass of an ion or the mass/charge ratio ofan ion refers to +/−0.50 atomic mass unit.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows LC-MS/MS profile of all analytes and metabolites.

FIG. 2 shows an example of baseline separation of (A) amitriptyline, (B)maprotiline, and (C) venlafaxine (analytes: left, internal standard(IS): right).

FIG. 3 shows accuracy of citalopram compared to another lab. Shows nobias greater than ±20% of values.

FIG. 4 shows accuracy of the metabolite desmethylcitalopram compared toanother lab. Shows no bias greater than ±20% of values.

FIG. 5 shows Cyclobenzaprine interference separated. The figure shows 5ng/mL Maprotiline+Cyclobenzaprine at 100×.

FIG. 6 shows mass spectral distinction of Desmethylvenlafaxine vs.Tramadol.

FIG. 7 shows Tramadol interference separated. The figure shows 5 ng/mLDesmethylvenlafaxine+Tramadol at 100×.

FIG. 8 shows Amitriptyline, Maprotiline, and Venlafaxine baselineseparation.

FIG. 9 shows Nortriptyline, Protriptyline, Desmethylvenlafaxine baselineseparation.

FIG. 10 shows Desmethyldoxapin and Mirtazapine baseline separation.

FIG. 11 shows Desipramine vs Mirtazapine Identified by differenttransitions.

FIG. 12 shows ion ratio and/or relative retention time (RRT) will failfor Desipramine in patients positive for Mirtazapine.

DETAILED DESCRIPTION OF THE INVENTION

In certain embodiments, antidepressant panels described herein can beused with compliance monitoring of patients having history/risk for useand or abuse of drugs within this class. Baseline testing, prior toprescribing this class of drugs, alerts the provider to the potentialfor polypharmacy drug conflicts. Compliance monitoring requiresprescribed drugs to be present and absence of non-prescribed drugs forthese patient populations.

Certain brain chemicals, neurotransmitters, are associated withdepression, more specifically serotonin, norepinephrine and dopamine.Most antidepressants treat depression by affecting theseneurotransmitters. Different types/classes of antidepressants affectthese neurotransmitters in different ways. These types include: SSRIs,SNRIs, NDRIs, Tricyclic, Atypical, MAOIs, and others (see below).

Selective serotonin reuptake inhibitors (SSRIs). Doctors often start byprescribing an SSRI. These medications are safer and generally causefewer bothersome side effects than other types of antidepressants. SSRIsinclude Fluoxetine (Prozac, Selfemra), Paroxetine (Paxil, Pexeva),Sertraline (Zoloft), Citalopram (Celexa), Escitalopram (Lexapro),Fluvoxamine (Faverin, Fevarin, Floxyfral, Dumyrox, Luvox), Vilazodone(Viibryd).

Serotonin and norepinephrine reuptake inhibitors (SNRIs)—duloxetine(Cymbalta), venlafaxine (Effexor XR), Desmethylvenlafaxine (syntheticform of venlafaxine's major metabolite, O-desmethylvenlafaxine; Pristiq,Khedezla) and levomilnacipran (Fetzima). SNRIs have unique dual actionin raising levels of both serotonin and norepinephrine; therefore SNRI'scombat more than one cause of depression.

Norepinephrine and dopamine reuptake inhibitors (NDRIs). Bupropion(Wellbutrin, Aplenzin, Forfivo XL) falls into this category. It's one ofthe few antidepressants not frequently associated with sexual sideeffects.

Tricyclic antidepressants (TCAs) tend to cause more side effects thannewer antidepressants. Tricyclic antidepressants generally aren'tprescribed unless the patient has tried an SSRI first withoutimprovement. TCAs include imipramine (Tofranil), nortriptyline(Pamelor), amitriptyline (Elavil, Endep, Lentizol, Levate, Saroten,Tryptanol, Tryptizol), Doxepin (Adapin, Curatin, Silenor, Sinequan),Trimipramine (Surmontil), Desipramine (Norpramin), Protriptyline(Vivactil), Amoxapine (Asendin), Clomipramine (Anafranil), andMaprotiline (Ludiomil).

Atypical antidepressants. These medications don't fit neatly into any ofthe other antidepressant categories. They include Trazodone (Oleptro),Mirtazapine (Remeron) and Vortioxetine (Brintellix). These are sedatingand usually taken in the evening.

Monoamine oxidase inhibitors (MAOIs) are not included in this assay.These medications can't be combined with SSRIs. Common MAOIs includetranylcypromine (Parnate), Phenelzine (Nardil) and Isocarboxazid(Marplan).

Methods are described for measuring the amount of analyte in a sample.More specifically, mass spectrometric methods are described fordetecting and/or quantifying analyte in a biological sample, such ashuman plasma or serum. The methods may utilize liquid chromatographyfollowed by tandem mass spectrometry to quantitate analyte in thesample.

Suitable test samples for use in methods of the present inventioninclude any test sample that may contain the analyte of interest. Insome preferred embodiments, a sample is a biological sample; that is, asample obtained from any biological source, such as an animal, a cellculture, an organ culture, etc. In certain preferred embodiments,samples are obtained from a mammalian animal, such as a dog, cat, horse,etc. Particularly preferred mammalian animals are primates, mostpreferably male or female humans. Preferred samples comprise bodilyfluids such as urine, blood, plasma, serum, saliva, cerebrospinal fluid,or tissue samples; preferably urine. Such samples may be obtained, forexample, from a patient; that is, a living person, male or female,presenting oneself in a clinical setting for diagnosis, prognosis, ortreatment of a disease or condition. In some embodiments, preferredsamples may be obtained from female humans of childbearing potential. Inembodiments where the sample comprises a biological sample, the methodsmay be used to determine the amount of leflunomide metabolite in thesample when the sample was obtained from the biological source (i.e.,the amount of endogenous leflunomide metabolite in the sample).

The present invention also contemplates kits for antidepressantquantitation assay. A kit for antidepressant quantitation assay mayinclude a kit comprising the compositions provided herein. For example,a kit may include packaging material and measured amounts of anisotopically labeled internal standard, in amounts sufficient for atleast one assay. Typically, the kits will also include instructionsrecorded in a tangible form (e.g., contained on paper or an electronicmedium) for using the packaged reagents for use in a antidepressantquantitation assay.

Calibration and QC pools for use in embodiments of the present inventionare preferably prepared using a matrix similar to the intended samplematrix, provided that analyte is essentially absent.

Sample Preparation for Mass Spectrometric Analysis

In preparation for mass spectrometric analysis, analytes may be enrichedrelative to one or more other components in the sample (e.g. protein) byvarious methods known in the art, including for example, liquidchromatography, filtration, centrifugation, thin layer chromatography(TLC), electrophoresis including capillary electrophoresis, affinityseparations including immunoaffinity separations, extraction methodsincluding ethyl acetate or methanol extraction, and the use ofchaotropic agents or any combination of the above or the like.

Protein precipitation is one method of preparing a test sample,especially a biological test sample, such as serum or plasma. Proteinpurification methods are well known in the art. For example, Polson etal., Journal of Chromatography B 2003, 785:263-275, describes proteinprecipitation techniques suitable for use in methods of the presentinvention. Protein precipitation may be used to remove most of theprotein from the sample leaving analytes in the supernatant. The samplesmay be centrifuged to separate the liquid supernatant from theprecipitated proteins; alternatively the samples may be filtered toremove precipitated proteins. The resultant supernatant or filtrate maythen be applied directly to mass spectrometry analysis; or alternativelyto additional purification methods, such as liquid chromatography, andsubsequent mass spectrometry analysis. In certain embodiments, the useof protein precipitation, such as for example, acetonitrile proteinprecipitation, may obviate the need for TFLC or other on-line extractionprior to mass spectrometry or high performance liquid chromatography(HPLC) and mass spectrometry.

Another method of sample purification that may be used prior to massspectrometry is liquid chromatography (LC). Certain methods of liquidchromatography, including high performance liquid chromatography (HPLC),rely on relatively slow, laminar flow technology. Traditional HPLCanalysis relies on column packing in which laminar flow of the samplethrough the column is the basis for separation of the analyte ofinterest from the sample. The skilled artisan will understand thatseparation in such columns is a partition process and may select LC,including HPLC, instruments and columns that are suitable for use withanalytes. The chromatographic column typically includes a medium (i.e.,a packing material) to facilitate separation of chemical moieties (i.e.,fractionation). The medium may include minute particles. The particlestypically include a bonded surface that interacts with the variouschemical moieties to facilitate separation of the chemical moieties. Onesuitable bonded surface is a hydrophobic bonded surface such as an alkylbonded, cyano bonded, or biphenyl bonded surface. Alkyl bonded surfacesmay include C-4, C-8, C-12, or C-18 bonded alkyl groups. In preferredembodiments, the column is a biphenyl column. The chromatographic columnincludes an inlet port for receiving a sample and an outlet port fordischarging an effluent that includes the fractionated sample. Thesample may be supplied to the inlet port directly, or from a SPE column,such as an on-line extraction column or a TFLC column. In someembodiments, an on-line guard cartridge may be used ahead of the HPLCcolumn to remove particulates and phospholipids in the samples prior tothe samples reaching the HPLC column. In some embodiments, guardcartridge may be a biphenyl guard cartridge.

In one embodiment, the sample may be applied to the LC column at theinlet port, eluted with a solvent or solvent mixture, and discharged atthe outlet port. Different solvent modes may be selected for eluting theanalyte(s) of interest. For example, liquid chromatography may beperformed using a gradient mode, an isocratic mode, or a polytypic (i.e.mixed) mode. During chromatography, the separation of materials iseffected by variables such as choice of eluent (also known as a “mobilephase”), elution mode, gradient conditions, temperature, etc.

In certain embodiments, an analyte may be purified by applying a sampleto a column under conditions where the analyte of interest is reversiblyretained by the column packing material, while one or more othermaterials are not retained. In these embodiments, a first mobile phasecondition can be employed where the analyte of interest is retained bythe column, and a second mobile phase condition can subsequently beemployed to remove retained material from the column, once thenon-retained materials are washed through. Alternatively, an analyte maybe purified by applying a sample to a column under mobile phaseconditions where the analyte of interest elutes at a differential ratein comparison to one or more other materials. Such procedures may enrichthe amount of one or more analytes of interest relative to one or moreother components of the sample.

In one preferred embodiment, HPLC is conducted with a biphenyl columnchromatographic system. In certain preferred embodiments, a biphenylanalytical column (e.g., a Pinnacle DB Biphenyl analytical column fromRestek Inc. (5 μm particle size, 50×2.1 mm), or equivalent) is used. Incertain preferred embodiments, HPLC is performed using HPLC Grade 0.1%aqueous formic acid as solvent A, and 0.1% formic acid in acetonitrileas solvent B.

By careful selection of valves and connector plumbing, two or morechromatography columns may be connected as needed such that material ispassed from one to the next without the need for any manual steps. Inpreferred embodiments, the selection of valves and plumbing iscontrolled by a computer pre-programmed to perform the necessary steps.Most preferably, the chromatography system is also connected in such anon-line fashion to the detector system, e.g., an MS system. Thus, anoperator may place a tray of samples in an autosampler, and theremaining operations are performed under computer control, resulting inpurification and analysis of all samples selected.

In some embodiments, TFLC may be used for purification of analytes priorto mass spectrometry. In such embodiments, samples may be extractedusing a TFLC column which captures the analyte. The analyte is theneluted and transferred on-line to an analytical HPLC column. Forexample, sample extraction may be accomplished with a TFLC extractioncartridge may be accomplished with a large particle size (50 μm) packedcolumn. Sample eluted off of this column is then transferred on-line toan HPLC analytical column for further purification prior to massspectrometry. Because the steps involved in these chromatographyprocedures may be linked in an automated fashion, the requirement foroperator involvement during the purification of the analyte can beminimized. This feature may result in savings of time and costs, andeliminate the opportunity for operator error.

Detection and Quantitation by Mass Spectrometry

In various embodiments, analytes may be ionized by any method known tothe skilled artisan. Mass spectrometry is performed using a massspectrometer, which includes an ion source for ionizing the fractionatedsample and creating charged molecules for further analysis. For example,ionization of the sample may be performed by electron ionization,chemical ionization, electrospray ionization (ESI), photon ionization,atmospheric pressure chemical ionization (APCI), photoionization,atmospheric pressure photoionization (APPI), laser diode thermaldesorption (LDTD), fast atom bombardment (FAB), liquid secondaryionization (LSI), matrix assisted laser desorption ionization (MALDI),field ionization, field desorption, thermospray/plasmaspray ionization,surface enhanced laser desorption ionization (SELDI), inductivelycoupled plasma (ICP) and particle beam ionization. The skilled artisanwill understand that the choice of ionization method may be determinedbased on the analyte to be measured, type of sample, the type ofdetector, the choice of positive versus negative mode, etc.

Analytes may be ionized in positive or negative mode. In someembodiments, analytes are ionized in positive mode.

In mass spectrometry techniques generally, after the sample has beenionized, the positively or negatively charged ions thereby created maybe analyzed to determine a mass to charge ratio (m/z). Suitableanalyzers for determining m/z include quadrupole analyzers, ion trapsanalyzers, and time-of-flight analyzers. Exemplary ion trap methods aredescribed in Bartolucci, et al., Rapid Commun. Mass Spectrom. 2000,14:967-73.

According to some methods of the present invention, high resolution/highaccuracy mass spectrometry is used for quantitation of analytes. Thatis, mass spectrometry is conducted with a mass spectrometer capable ofexhibiting a resolving power (FWHM) of at least 10,000, with accuracy ofabout 50 ppm or less for the ions of interest; preferably the massspectrometer exhibits a resolving power (FWHM) of 20,000 or better andaccuracy of about 20 ppm or less; such as a resolving power (FWHM) of25,000 or better and accuracy of about 5 ppm or less; such as aresolving power (FWHM) of 25,000 or better and accuracy of about 3 ppmor less. Three exemplary mass spectrometers capable of exhibiting therequisite level of performance for analyte ions are those which includeorbitrap mass analyzers, certain TOF mass analyzers, or Fouriertransform ion cyclotron resonance mass analyzers.

Elements found in biological active molecules, such as carbon, oxygen,and nitrogen, naturally exist in a number of different isotopic forms.For example, most carbon is present as ¹²C, but approximately 1% of allnaturally occurring carbon is present as ¹³C. Thus, some fraction ofnaturally occurring carbon containing molecules will contain at leastone ¹³C atom. Inclusion of naturally occurring elemental isotopes inmolecules gives rise to multiple molecular isotopic forms. Thedifference in masses of molecular isotopic forms is at least 1 atomicmass unit (amu). This is because elemental isotopes differ by at leastone neutron (mass of one neutron≈1 amu). When molecular isotopic formsare ionized to multiply charged states, the mass distinction between theisotopic forms can become difficult to discern because massspectrometric detection is based on the mass to charge ratio (m/z). Forexample, two isotopic forms differing in mass by 1 amu that are bothionized to a 5+ state will exhibit differences in their m/z of only 0.2(difference of 1 amu/charge state of 5). High resolution/high accuracymass spectrometers are capable of discerning between isotopic forms ofhighly multiply charged ions (such as ions with charges of ±4, ±5, ±6,±7, ±8, ±9, or higher).

Due to naturally occurring elemental isotopes, multiple isotopic formstypically exist for every molecular ion (each of which may give rise toa separately detectable spectrometric peak if analyzed with a sensitiveenough mass spectrometric instrument). The m/z ratios and relativeabundances of multiple isotopic forms collectively comprise an isotopicsignature for a molecular ion. In some embodiments, the m/z and relativeabundances of two or more molecular isotopic forms may be utilized toconfirm the identity of a molecular ion under investigation. In someembodiments, the mass spectrometric peak from one or more isotopic formsis used to quantitate a molecular ion. In some related embodiments, asingle mass spectrometric peak from one isotopic form is used toquantitate a molecular ion. In other related embodiments, a plurality ofisotopic peaks are used to quantitate a molecular ion. In these laterembodiments, the plurality of isotopic peaks may be subject to anyappropriate mathematical treatment. Several mathematical treatments areknown in the art and include, but are not limited to summing the areaunder multiple peaks or averaging the response from multiple peaks.

In mass spectrometry techniques generally, ions may be detected usingseveral detection modes. For example, selected ions may be detected,i.e. using a selective ion monitoring mode (SIM), or alternatively, masstransitions resulting from collision activated dissociation (CAD), e.g.,multiple reaction monitoring (MRM) or selected reaction monitoring(SRM). CAD is often used to generate fragment ions for furtherdetection. In CAD, precursor ions gain energy through collisions with aninert gas, and subsequently fragment by a process referred to as“unimolecular decomposition.” Sufficient energy must be deposited in theprecursor ion so that certain bonds within the ion can be broken due toincreased vibrational energy. Alternatively, neutral loss may bemonitored.

In some embodiments, the mass-to-charge ratio is determined using aquadrupole analyzer. For example, in a “quadrupole” or “quadrupole iontrap” instrument, ions in an oscillating radio frequency fieldexperience a force proportional to the DC potential applied betweenelectrodes, the amplitude of the RF signal, and the mass/charge ratio.The voltage and amplitude may be selected so that only ions having aparticular mass/charge ratio travel the length of the quadrupole, whileall other ions are deflected. Thus, quadrupole instruments may act asboth a “mass filter” and as a “mass detector” for the ions injected intothe instrument.

One may enhance the specificity of the MS technique by employing “tandemmass spectrometry,” or “MS/MS”. In this technique, a precursor ion (alsocalled a parent ion) generated from a molecule of interest can befiltered in an MS instrument, and the precursor ion subsequentlyfragmented to yield one or more fragment ions (also called daughter ionsor product ions) that are then analyzed in a second MS procedure. Bycareful selection of precursor ions, only ions produced by certainanalytes are passed to the fragmentation chamber, where collisions withatoms of an inert gas produce the fragment ions. Because both theprecursor and fragment ions are produced in a reproducible fashion undera given set of ionization/fragmentation conditions, the MS/MS techniquemay provide an extremely powerful analytical tool. For example, thecombination of filtration/fragmentation may be used to eliminateinterfering substances, and may be particularly useful in complexsamples, such as biological samples.

Alternate modes of operating a tandem mass spectrometric instrumentinclude product ion scanning and precursor ion scanning. For adescription of these modes of operation, see, e.g., E. Michael Thurman,et al., Chromatographic-Mass Spectrometric Food Analysis for TraceDetermination of Pesticide Residues, Chapter 8 (Amadeo R.Fernandez-Alba, ed., Elsevier 2005) (387).

The results of an analyte assay may be related to the amount of theanalyte in the original sample by numerous methods known in the art. Forexample, given that sampling and analysis parameters are carefullycontrolled, the relative abundance of a given ion may be compared to atable that converts that relative abundance to an absolute amount of theoriginal molecule. Alternatively, external standards may be run with thesamples, and a standard curve constructed based on ions generated fromthose standards. Using such a standard curve, the relative abundance ofa given ion may be converted into an absolute amount of the originalmolecule. In certain preferred embodiments, an internal standard is usedto generate a standard curve for calculating the quantity of analytes.Methods of generating and using such standard curves are well known inthe art and one of ordinary skill is capable of selecting an appropriateinternal standard. For example, one or more forms of an isotopicallylabeled molecule with a similar m/z as analytes may be used as internalstandards. In some embodiments described herein, an exemplary internalstandard is an isotopically labeled diazepam, although numerous othercompounds (isotopically labeled or otherwise) may be used. Numerousother methods for relating the amount of an ion to the amount of theoriginal molecule will be well known to those of ordinary skill in theart.

As used herein, an “isotopic label” produces a mass shift in the labeledmolecule relative to the unlabeled molecule when analyzed by massspectrometric techniques. Examples of suitable labels include deuterium(²H), ¹³C, and ¹⁵N. One or more isotopic labels can be incorporated atone or more positions in the molecule and one or more kinds of isotopiclabels can be used on the same isotopically labeled molecule.

One or more steps of the methods may be performed using automatedmachines. In certain embodiments, one or more purification steps areperformed on-line, and more preferably all of the purification and massspectrometry steps may be performed in an on-line fashion.

In particularly preferred embodiments, analytes in a sample are detectedand/or quantified using MS/MS as follows. Samples are preferablysubjected to liquid chromatography, preferably HPLC; the flow of liquidsolvent from a chromatographic column enters the heated nebulizerinterface of an MS/MS analyzer; and the solvent/analyte mixture isconverted to vapor in the heated charged tubing of the interface. Duringthese processes, the analyte (i.e., antidepressants or metabolites) isanalyzed. The ions, e.g. precursor ions, pass through the orifice of theinstrument and enter the first quadrupole. Quadrupoles 1 and 3 (Q1 andQ3) are mass filters, allowing selection of ions (i.e., selection of“precursor” and “fragment” ions in Q1 and Q3, respectively) based ontheir mass to charge ratio (m/z). Quadrupole 2 (Q2) is the collisioncell, where ions are fragmented. The first quadrupole of the massspectrometer (Q1) selects for molecules with the mass to charge ratiosof analytes. Precursor ions with the correct mass/charge ratios areallowed to pass into the collision chamber (Q2), while unwanted ionswith any other mass/charge ratio collide with the sides of thequadrupole and are eliminated. Precursor ions entering Q2 collide withneutral argon gas molecules and fragment. The fragment ions generatedare passed into quadrupole 3 (Q3), where the fragment ions of analytesare selected while other ions are eliminated.

The methods may involve MS/MS performed in either positive or negativeion mode; preferably positive ion mode. Using standard methods wellknown in the art, one of ordinary skill is capable of identifying one ormore fragment ions of a particular precursor ion of analytes that may beused for selection in quadrupole 3 (Q3).

As ions collide with the detector they produce a pulse of electrons thatare converted to a digital signal. The acquired data is relayed to acomputer, which plots counts of the ions collected versus time. Theresulting mass chromatograms are similar to chromatograms generated intraditional HPLC-MS methods. The areas under the peaks corresponding toparticular ions, or the amplitude of such peaks, may be measured andcorrelated to the amount of the analyte of interest. In certainembodiments, the area under the curves, or amplitude of the peaks, forfragment ion(s) and/or precursor ions are measured to determine theamount of analytes. As described above, the relative abundance of agiven ion may be converted into an absolute amount of the originalanalyte using calibration standard curves based on peaks of one or moreions of an internal or external molecular standard.

The following Examples serve to illustrate the invention. These Examplesare in no way intended to limit the scope of the methods.

EXAMPLES Example 1 Sample Preparation

We describe a validated LC-MS/MS method for simultaneous analysis for 23prescribed antidepressant analytes and their metabolites provided inTable 1 below.

TABLE 1 Antidepressants and metabolites determined by the assay NameClass Brand Names Amitriptyline TCA Elavil, Endep, Lentizol, Levate,Saroten, Tryptanol, Tryptizol Amoxapine TCA AsendinCitalopram/Escitalopram SSRI Celexa, LexaproDesmethylcitalopram—METABOLITE Clomipramine TCA AnafranilDesmethylclomipramine—METABOLITE Desipramine TCA Norpramin Doxepin TCAAdapin, Curatin, Silenor, Sinequan Desmethyldoxepin—METABOLITEDuloxetine SNRI Cymbalta Fluoxetine SSRI Prozac, SelfemraNorfluoxetine—METABOLITE Fluvoxamine SSRI Faverin, Fevarin, Floxyfral,Dumyrox, Luvox Norfluvoxamine—METABOLITE Hydroxybupropion NDRIWellbutrin, Aplenzin, Forfivo XL Imipramine TCA Tofranil Maprotiline TCALudiomil Mirtazapine Atypical Remeron Nortriptyline—METABOLITE of TCAPamelor Amitriptyline & Prescribed Drug Paroxetine SSRI Paxil, PexevaProtriptyline TCA Vivactil Sertraline SSRI ZoloftNorsertraline—METABOLITE Trazodone Atypical Oleptro1,3-chlorphenylpiperazine (metaCPP)—METABOLITE Trimipramine TCASurmontil Venlafaxine SNRI Effexor XR O-Desmethylvenlafaxine—METABOLITESNRI Pristiq, Khedezla & Prescribed Drug Vilazodone SSRI ViibrydVortioxetine Atypical Brintellix

Quality Controls, Calibrators, and Internal Standards: Calibrationstandards (4-5,000 ng/mL) and quality controls (QC's) at 5, 12.5, and4,000 ng/mL were prepared by spiking stock solutions of analytes intodrug-free urine controls (UTAK). The internal standard (IS) was a 25-100ng/mL mixture of 1,3-chlorphenylpiperazine-D8, hydroxybupropion-D6,desmethyl-venlafaxine-D6, desmethylcitalopram-D3, trimipramine-D3,amitriptyline-D3, nortriptyline-D3, paroxetine-D6, protriptyline-D3,citalopram-D6, venlafaxine-D6, imipramine-D3, trazodone-D6,vilazodone-D4, and vortioxetine-D8.

Sample Preparation: Urine samples, calibrators, and QCs (25 μL each)were mixed with IS (25 μL) in a 1 mL, 96 well extraction plate, dilutedwith 450 μL of 10 mM ammonium formate in water (mobile phase A), andthen vortexed at 1,100 rpm for 2 minutes before being moved to theLC-MS/MS for injection and analysis.

Example 2 Liquid Chromatography-Mass Spectrometry

LC-MS/MS: Extracted samples (25 μL) were chromato-graphically resolvedon a Kinetex® Phenyl-Hexyl 50×4.6 mm 2.6 μcolumn (Phenomenex) usingmobile phase A/mobile phase B (25% methanol in acetonitrile) gradients.A 4-column LC multiplex was employed to maximize throughput on a PreludeLX-4 MD™ (ThermoFisher Scientific). A Sciex 4500 Triple Quad™ MassSpectrometer was used for selected reaction monitoring. FIG. 1 is arepresentative chromatogram for all analytes, and FIG. 2 demonstratesbaseline separation of closely related analytes.

Table 2 provides the mass transitions used to detect each analyte in themass spectrometry assay.

TABLE 2 Mass spectrometry transitions (m/z) used for detectingantidepressants and metabolites Q1 (m/z) Q3 (m/z) ID DP EP CE CXP 1196.993 118 1,3-Chlorphenylpiperazine 1 76 5 60 8 2 196.993 119.11,3-Chlorphenylpiperazine 2 76 5 45 10 3 205.065 158.11,3-Chlorphenylpiperazine D8 56 10 29 12 4 278.096 105 Amitriptyline 181 10 50 10 5 278.096 115 Amitriptyline 2 81 10 95 10 6 281 202.1Amitriptyline-D3 81 10 79 14 7 314.006 271.1 Amoxapine 1 101 10 48 10 8314.006 193.1 Amoxapine 2 101 10 78 14 9 256.02 130 Hydroxybupropion 156 10 85 12 10 256.02 103 Hydroxybupropion 2 56 10 53 10 11 262.061130.1 Hydroxybupropion-D6 1 10 65 10 12 325.07 109 Citalopram 1 81 10 9010 13 325.07 262.1 Citalopram 2 81 10 39 10 14 331.103 109 Citalopram-D6 76 10 33 10 15 264.083 91 Nortriptyline 1 76 10 70 8 16 264.083 105Nortriptyline 2 76 10 45 10 17 267.095 105 Nortriptyline-D3 71 10 27 1018 311.043 109 Desmethylcitalopram 1 81 10 85 10 19 311.043 262.1Desmethylcitalopram 2 81 10 36 10 20 314.072 108.9Desmethylcitalopram-D3 81 10 31 10 21 315.054 86.1 Clomipramine 1 81 1055 8 22 315.054 58 Clomipramine 2 81 10 30 16 23 301.037 72Desmethylclomipramine 1 76 10 60 8 24 301.037 227.1Desmethylclomipramine 2 76 10 51 16 25 267.091 72 Desipramine 1 71 5 5510 26 267.091 193.1 Desipramine 2 71 5 60 14 27 280.096 107 Doxepin 1 7610 55 10 28 280.096 165.1 Doxepin 2 76 5 95 14 29 266.074 107Desmethyldoxapin 1 66 10 52 10 30 266.074 77 Desmethyldoxapin 2 66 10 908 31 298.03 154.1 Duloxetine 1 11 5 8 8 32 298.03 44.1 Duloxetine 2 11 590 9 33 310.07 148.1 Fluoxetine 1 16 5 13 10 34 310.07 44.1 Fluoxetine 216 5 13 9 35 296.066 134.2 Norfluoxetine 1 17 5 13 10 36 296.066 30.1Norfluoxetine 2 10 5 35 8 37 319.057 71 Fluvoxamine 1 16 5 33 8 38319.057 200.1 Fluvoxamine 2 16 5 31 8 39 305.025 229.1 Norfluvoxamine 11 5 23 10 40 305.025 188.1 Norfluvoxamine 2 1 5 27 8 41 281.098 86Imipramine 1 66 10 60 8 42 281.098 58 Imipramine 2 66 10 115 6 43284.013 89 Imipramine-D3 66 10 21 8 44 278.094 191.2 Maprotiline 1 81 1065 8 45 278.094 189 Maprotiline 2 81 10 105 14 46 266.081 195.1Mirtazapine 1 86 10 60 14 47 266.081 194.1 Mirtazapine 2 135 10 67 7 48330.033 192.1 Paroxetine 1 106 10 29 8 49 330.033 70 Paroxetine 2 106 1070 8 50 336.092 198.2 Paroxetine-D6 71 10 29 8 51 264.096 191Protriptyline 1 81 10 55 16 52 264.096 189 Protriptyline 2 81 10 95 1453 267.095 191.1 Protriptyline-D3 86 10 39 14 54 306 159 Sertraline 1 665 39 10 55 306 275 Sertraline 2 66 5 17 12 56 292.005 159Desmethylsertraline 1 6 5 35 8 57 292.005 123 Desmethylsertraline 2 6 567 10 58 372.096 176.1 Trazodone 1 111 5 45 12 59 372.096 148 Trazodone2 111 5 70 12 60 378.114 182.1 Trazodone-D6 116 10 33 14 61 295.128100.1 Trimipramine 1 1 10 55 10 62 295.128 58.1 Trimipramine 2 1 10 1156 63 298.138 103.1 Trimipramine-D3 31 10 23 10 64 278.126 58 Venlafaxine1 1 10 90 16 65 278.126 121 Venlafaxine 2 1 10 60 12 66 284.139 64.1Venlafaxine-D6 1 10 57 6 67 264.106 58 Desmethylvenlafaxine 1 1 10 75 668 264.106 107 Desmethylvenlafaxine 2 1 10 65 10 69 270.134 64.1Desmethylvenlafaxine-D6 1 10 49 6 70 442.133 155.1 Vilazodone 1 151 5 9512 71 442.133 197.2 Vilazodone 2 151 5 45 8 72 446.163 155.1Vilazodone-D4 151 5 69 12 73 299.059 150 Vortioxetine 1 126 10 44 7 74299.059 109 Vortioxetine 2 126 10 44 7 75 307.082 153.1 Vortioxetine-D8101 5 38 8 278.2 202.1 Amitriptyline 3 50 10 35 5 310.1 117.1 Fluoxetine3 50 10 35 5 310.1 91.1 Fluoxetine 4 50 10 35 5 310.1 259.1 Fluoxetine 550 10 35 5 319.2 145.1 Fluvoxamine 3 50 10 35 5 319.2 130.1 Fluvoxamine4 50 10 35 5 266.2 209.2 Mirtazapine 3 50 10 35 5 330.1 135.1 Paroxetine3 50 10 35 5 330.1 109 Paroxetine 4 50 10 35 5 264.2 155.2 Protriptyline3 50 10 35 5 264.2 178.2 Protriptyline 4 50 10 35 5 306 129.1 Sertraline3 50 10 35 5 295.2 193.1 Trimipramine 3 50 10 35 5 295.2 208.2Trimipramine 4 50 10 35 5 278.2 147.1 Venlafaxine 3 50 10 35 5 278.291.1 Venlafaxine 4 50 10 35 5 278.2 191.1 Amitriptyline 4 50 10 35 5

Example 3 Validation and Results

Validation: The following characteristics were determined by standardlaboratory methods: limit of quantification (LOQ), linearity (includingupper limit of linearity [ULOL] with dilution), precision, accuracy,interference by over 150 different drugs, stability, extracted specimenstability, matrix effect, and carryover.

Linearity:

A 5-9 point calibration curve exhibited consistent linearity andreproducibility in the range of ±20% of their target with regressioncoefficient (r)>0.990.

The CVs were between 7.5% and 10%.

The analytical measurement range (AMR) for all antidepressants analytesand metabolites was 4 to 5,000 ng/mL, with an LOQ of 10 ng/ml (with 1exception), and an ULOL of 50,000 ng/mL. The exception was themetabolite, norsertraline, with an AMR of 25 to 5,000 ng/mL and a LOQ of50 ng/mL.

Precision:

A precision study over a 5 day period showed consistent results with asigma value of greater than 3 for the low, middle and high level QC's.

Accuracy:

The accuracy study was carried out by correlating 65 sample across theconcentration range of 4 to 20,000 ng/mL with another 65 results fromanother laboratory. Examples are presented in FIGS. 3 and 4 .

On average, Deming regression showed a correlation coefficient of 1.022and an intercept of −0.0681 with no bias.

Interference:

(Over 150 multiple illicit drugs and prescription drugs at 100 times thecutoff was tested. These tests were done using both in negative matrixcontrol and LoQ control spiked with the relevant substances.)

None of the interference drugs tested cause ≥20% deviation in the signalintensities of the panel drugs at the LOQ.

Stability:

Specimens were stable for 7 days at room temperature, 14 daysrefrigerated, and 30 days frozen. Post-extraction, samples were stablefor 24 hrs.

Matrix Effects:

Samples were compared at 3 different levels (0.5×, 2×, and 0.8×ULOL)with neat and diluted matrixes.

No matrix effects were observed.

Carryover:

Two samples were spiked at 4000 ng/mL back to back followed by 4 blanksamples to determine carryover effects. This was ran in triplicate.

No carryover was observed.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the invention embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods. This includes the genericdescription of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

That which is claimed is:
 1. A method for detecting or determining theamount of one or more selective serotonin reuptake inhibitors (SSRI) andSSRI metabolites in a sample by tandem mass spectrometry, said methodcomprising: a. subjecting the sample to ionization under conditionssuitable to produce one or more ions detectable by mass spectrometry; b.determining the amount of one or more ions by tandem mass spectrometry,wherein the one or more ions comprise a fluoxetine fragment ion with amass/charge ratio of 148.1±0.2 and a norfluoxetine fragment ion with amass/charge ratio of 134.2±0.2 or 30.1±0.2; and c. using the amount ofthe one or more ions determined in step (b) to determine the amount ofSSRI and SSRI metabolites comprising fluoxetine and norfluoxetine in thesample.
 2. The method of claim 1, wherein the method comprisessimultaneously detecting or determining the amount of 10 or more SSRIand SSRI metabolites.
 3. The method of claim 1, wherein one or moreinternal standards are added.
 4. The method of claim 3, wherein the oneor more internal standards comprise deuterated internal standards. 5.The method of claim 4, wherein the deuterated internal standards areselected from the group consisting of desmethylcitalopram-D3,paroxetine-D6, citalopramD6, and vilazodone-D4.
 6. The method of claim1, wherein the sample comprises a biological sample.
 7. The method ofclaim 1, wherein the sample comprises urine.
 8. The method of claim 1,wherein the sample is subjected to liquid chromatography prior toionization.
 9. The method of claim 8, wherein said liquid chromatographycomprises high performance liquid chromatography.
 10. The method ofclaim 1, wherein the method is capable of detecting SSRI and SSRImetabolites at levels within the range of about 4 ng/mL to about 5000ng/mL, inclusive.
 11. The method of claim 1, wherein the method iscapable of detecting SSRI and SSRI metabolites at levels within therange of about 25 ng/mL to about 5000 ng/mL, inclusive.
 12. The methodof claim 1, wherein said tandem mass spectrometry is conducted byselected reaction monitoring, multiple reaction monitoring, precursorion scanning, or product ion scanning.
 13. The method of claim 1,wherein said tandem mass spectrometry is conducted by selected reactionmonitoring.
 14. The method of claim 1, wherein the lower limit ofquantitation is 10 ng/mL.
 15. The method of claim 1, wherein the lowerlimit of quantitation is 50 ng/mL.
 16. The method of claim 1, whereinthe sample comprises serum.